Bottom Line:
The deduced receptor proteins are the first descriptions of H. americanus neuropeptide receptors, and include ones for most of the peptide groups mentioned earlier, as well as those for ecdysis-triggering hormone, red pigment concentrating hormone and short neuropeptide F.Multiple receptors were identified for most peptide families.These data represent the most complete description of the molecular underpinnings of peptidergic signaling in H. americanus, and will serve as a foundation for future gene-based studies of neuropeptidergic control in the lobster.

Affiliation: Békésy Laboratory of Neurobiology, Pacific Biosciences Research Center and Technology, 6500 College Station, University of Hawaii at Manoa, 1993 East-West Road, Honolulu, Hawaii, 96822, United States of America.

ABSTRACTPeptides are the largest and most diverse class of molecules used for neurochemical communication, playing key roles in the control of essentially all aspects of physiology and behavior. The American lobster, Homarus americanus, is a crustacean of commercial and biomedical importance; lobster growth and reproduction are under neuropeptidergic control, and portions of the lobster nervous system serve as models for understanding the general principles underlying rhythmic motor behavior (including peptidergic neuromodulation). While a number of neuropeptides have been identified from H. americanus, and the effects of some have been investigated at the cellular/systems levels, little is currently known about the molecular components of neuropeptidergic signaling in the lobster. Here, a H. americanus neural transcriptome was generated and mined for sequences encoding putative peptide precursors and receptors; 35 precursor- and 41 receptor-encoding transcripts were identified. We predicted 194 distinct neuropeptides from the deduced precursor proteins, including members of the adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin C, bursicon, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptide, diuretic hormone 31, diuretic hormone 44, eclosion hormone, FLRFamide, GSEFLamide, insulin-like peptide, intocin, leucokinin, myosuppressin, neuroparsin, neuropeptide F, orcokinin, pigment dispersing hormone, proctolin, pyrokinin, SIFamide, sulfakinin and tachykinin-related peptide families. While some of the predicted peptides are known H. americanus isoforms, most are novel identifications, more than doubling the extant lobster neuropeptidome. The deduced receptor proteins are the first descriptions of H. americanus neuropeptide receptors, and include ones for most of the peptide groups mentioned earlier, as well as those for ecdysis-triggering hormone, red pigment concentrating hormone and short neuropeptide F. Multiple receptors were identified for most peptide families. These data represent the most complete description of the molecular underpinnings of peptidergic signaling in H. americanus, and will serve as a foundation for future gene-based studies of neuropeptidergic control in the lobster.

pone.0145964.g003: Amino acid sequence alignment of selected Homarus americanus receptors for peptide families in which multiple full-length receptors were identified.(A) Alignment of allatostatin-C receptor (AST-CR) I and II. (B) Alignment of pigment dispersing hormone receptor (PDHR) I and II. In each panel, “*” located beneath each line of the alignment indicates residues that are identical in the two sequences, while “:” and “.” indicate highly conservative and conservative substituted (similar) amino acids, respectively, shared between the two proteins. In this figure, the rhodopsin-like G protein-coupled receptor (GPCR) seven transmembrane domains identified by InterPro in each of the two AST-CRs are highlighted in black, while the GPCR family 2 extracellular hormone receptor domain and the GPCR family 2-like seven transmembrane region identified by InterPro in the each PDHR are highlighted in red and dark blue, respectively.

Mentions:
InterPro analysis of the AST-A (Fig 2A), AST-C I (Fig 3A), AST-C II (Fig 3A), CCHamide I, CCHamide II, ETH I, ETH II, FLRFamide, leucokinin, myosuppressin, NPF I, NPF II, NPF III, NPF IV, proctolin I, sNPF, SIFamide, sulfakinin and TRP I receptors placed each of these proteins into the rhodopsin-like G protein-coupled receptor (GPCR) superfamily (InterPro ID No.IPR000276), with each protein predicted to possess a single rhodopsin-like GPCR seven transmembrane domain (InterPro ID No.IPR017452) (highlighted in black in Figs 2A and 3A and in S4 Fig). Analyses of the DH31 I, DH44 I (Fig 2B), PDH I (Fig 3B) and PDH II (Fig 3B) receptors using InterPro identified these proteins as members of the secretin-like GPCR family 2 superfamily (InterPro ID No.IPR000832). A single GPCR family 2 extracellular hormone receptor domain (InterPro ID No. IPR001879) (highlighted in red in Figs 2B and 3B, and in S4 Fig) and a single GPCR family 2-like seven transmembrane region (InterPro ID No.IPR017981) (highlighted in dark blue in Figs 2B and 3B, and in S4 Fig) was identified in each of these receptors. No protein family membership was identified by InterPro for the ILP I receptor (Fig 2C and S4 Fig). However, analysis using this program did predict a number of functional regions within this receptor’s sequence, including two receptor L-domains (InterPro ID No.IPR000494) (highlighted in light blue and green in Fig 2C and S4 Fig), one furin-like cysteine-rich domain (InterPro ID No.IPR006211) (highlighted in green and yellow in Fig 2C and S4 Fig), two immunoglobulin-like fold (fibronectin type III subtype [InterPro ID No.IPR003961]) domains (InterPro ID No.IPR013783) (highlighted in pink in Fig 2C and S4 Fig), and one protein kinase-like (tyrosine-protein kinase, catalytic domain subtype [InterPro ID No.IPR020635]) domain (InterPro ID No.IPR011009) (highlighted in gray in Fig 2C and S4 Fig). Interestingly, in the type-1 insulin-like growth-factor receptor, the first three domains of this protein’s extracellular portion consist of two L-domains and a single cysteine rich region, which are hypothesized to form a binding pocket for insulin [90]; a similar situation may be at play in the lobster ILP I receptor identified here.

pone.0145964.g003: Amino acid sequence alignment of selected Homarus americanus receptors for peptide families in which multiple full-length receptors were identified.(A) Alignment of allatostatin-C receptor (AST-CR) I and II. (B) Alignment of pigment dispersing hormone receptor (PDHR) I and II. In each panel, “*” located beneath each line of the alignment indicates residues that are identical in the two sequences, while “:” and “.” indicate highly conservative and conservative substituted (similar) amino acids, respectively, shared between the two proteins. In this figure, the rhodopsin-like G protein-coupled receptor (GPCR) seven transmembrane domains identified by InterPro in each of the two AST-CRs are highlighted in black, while the GPCR family 2 extracellular hormone receptor domain and the GPCR family 2-like seven transmembrane region identified by InterPro in the each PDHR are highlighted in red and dark blue, respectively.

Mentions:
InterPro analysis of the AST-A (Fig 2A), AST-C I (Fig 3A), AST-C II (Fig 3A), CCHamide I, CCHamide II, ETH I, ETH II, FLRFamide, leucokinin, myosuppressin, NPF I, NPF II, NPF III, NPF IV, proctolin I, sNPF, SIFamide, sulfakinin and TRP I receptors placed each of these proteins into the rhodopsin-like G protein-coupled receptor (GPCR) superfamily (InterPro ID No.IPR000276), with each protein predicted to possess a single rhodopsin-like GPCR seven transmembrane domain (InterPro ID No.IPR017452) (highlighted in black in Figs 2A and 3A and in S4 Fig). Analyses of the DH31 I, DH44 I (Fig 2B), PDH I (Fig 3B) and PDH II (Fig 3B) receptors using InterPro identified these proteins as members of the secretin-like GPCR family 2 superfamily (InterPro ID No.IPR000832). A single GPCR family 2 extracellular hormone receptor domain (InterPro ID No. IPR001879) (highlighted in red in Figs 2B and 3B, and in S4 Fig) and a single GPCR family 2-like seven transmembrane region (InterPro ID No.IPR017981) (highlighted in dark blue in Figs 2B and 3B, and in S4 Fig) was identified in each of these receptors. No protein family membership was identified by InterPro for the ILP I receptor (Fig 2C and S4 Fig). However, analysis using this program did predict a number of functional regions within this receptor’s sequence, including two receptor L-domains (InterPro ID No.IPR000494) (highlighted in light blue and green in Fig 2C and S4 Fig), one furin-like cysteine-rich domain (InterPro ID No.IPR006211) (highlighted in green and yellow in Fig 2C and S4 Fig), two immunoglobulin-like fold (fibronectin type III subtype [InterPro ID No.IPR003961]) domains (InterPro ID No.IPR013783) (highlighted in pink in Fig 2C and S4 Fig), and one protein kinase-like (tyrosine-protein kinase, catalytic domain subtype [InterPro ID No.IPR020635]) domain (InterPro ID No.IPR011009) (highlighted in gray in Fig 2C and S4 Fig). Interestingly, in the type-1 insulin-like growth-factor receptor, the first three domains of this protein’s extracellular portion consist of two L-domains and a single cysteine rich region, which are hypothesized to form a binding pocket for insulin [90]; a similar situation may be at play in the lobster ILP I receptor identified here.

Bottom Line:
The deduced receptor proteins are the first descriptions of H. americanus neuropeptide receptors, and include ones for most of the peptide groups mentioned earlier, as well as those for ecdysis-triggering hormone, red pigment concentrating hormone and short neuropeptide F.Multiple receptors were identified for most peptide families.These data represent the most complete description of the molecular underpinnings of peptidergic signaling in H. americanus, and will serve as a foundation for future gene-based studies of neuropeptidergic control in the lobster.

Affiliation:
Békésy Laboratory of Neurobiology, Pacific Biosciences Research Center and Technology, 6500 College Station, University of Hawaii at Manoa, 1993 East-West Road, Honolulu, Hawaii, 96822, United States of America.

ABSTRACTPeptides are the largest and most diverse class of molecules used for neurochemical communication, playing key roles in the control of essentially all aspects of physiology and behavior. The American lobster, Homarus americanus, is a crustacean of commercial and biomedical importance; lobster growth and reproduction are under neuropeptidergic control, and portions of the lobster nervous system serve as models for understanding the general principles underlying rhythmic motor behavior (including peptidergic neuromodulation). While a number of neuropeptides have been identified from H. americanus, and the effects of some have been investigated at the cellular/systems levels, little is currently known about the molecular components of neuropeptidergic signaling in the lobster. Here, a H. americanus neural transcriptome was generated and mined for sequences encoding putative peptide precursors and receptors; 35 precursor- and 41 receptor-encoding transcripts were identified. We predicted 194 distinct neuropeptides from the deduced precursor proteins, including members of the adipokinetic hormone-corazonin-like peptide, allatostatin A, allatostatin C, bursicon, CCHamide, corazonin, crustacean cardioactive peptide, crustacean hyperglycemic hormone (CHH), CHH precursor-related peptide, diuretic hormone 31, diuretic hormone 44, eclosion hormone, FLRFamide, GSEFLamide, insulin-like peptide, intocin, leucokinin, myosuppressin, neuroparsin, neuropeptide F, orcokinin, pigment dispersing hormone, proctolin, pyrokinin, SIFamide, sulfakinin and tachykinin-related peptide families. While some of the predicted peptides are known H. americanus isoforms, most are novel identifications, more than doubling the extant lobster neuropeptidome. The deduced receptor proteins are the first descriptions of H. americanus neuropeptide receptors, and include ones for most of the peptide groups mentioned earlier, as well as those for ecdysis-triggering hormone, red pigment concentrating hormone and short neuropeptide F. Multiple receptors were identified for most peptide families. These data represent the most complete description of the molecular underpinnings of peptidergic signaling in H. americanus, and will serve as a foundation for future gene-based studies of neuropeptidergic control in the lobster.